DESCRIPTION: The development of a general approach for the site-specific incorporation of unnatural amino acids into proteins in vivo, directly from the growth media, would greatly enhance the power of unnatural amino acid mutagenesis. For example, the ability to synthesize large quantities of proteins containing heavy atoms would facilitate protein structure determination, and the ability to site-selectively substitute fluorophores or photocleavable groups into proteins in living cells would provide powerful tools for studying protein function in vivo. Alternatively, one might be able to enhance the properties of proteins by providing building blocks with new functional groups, such as a keto-containing amino acid. To this end, the Schultz group has developed a general multistage approach for site specifically incorporating unnatural amino acids into proteins in vivo. The overall strategy consists of four elements: (1) The design and synthesis of the unnatural amino acid substrate; (2) The construction of an orthogonal amber suppressor tRNA (O-tRNACUA) that is not the substrate for any of the naturally occurring aminoacyl tRNA synthetases and which will eventually be used to deliver the unnatural amino acid in response to a UAG codon in the mRNA encoding the protein of interest; (3) the selection of aminoacyl tRNA synthase from a library of mutants that recognizes the O-tRNACUA but does not recognize any endogenous tRNAs; and (4) the screening of a library of mutants of this tRNA aminoacyl synthetase for the ability to acylate the O-tRNACUA with the unnatural amino acid but not a common amino acid.